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Abstract:

Substance productivity is improved by introducing a metabolic pathway for
synthesis of acetyl-CoA or acetic acid from glucose-6-phosphate into
yeast. Acetic acid productivity, acetyl-CoA productivity, and
productivity of a substance made from acetyl-CoA-derived are improved by
attenuating genes involved in the glycolytic system of yeast and
introducing a phosphoketolase gene into the yeast.

Claims:

1. (canceled)

2. A recombinant yeast, which comprises an attenuated phosphofructokinase
gene and an introduced phosphoketolase gene, wherein the expression of a
glucose-6-phosphate dehydrogenase gene and/or a
D-ribulose-5-phosphate-3-epimerase gene is enhanced.

3. A recombinant yeast, which comprises an attenuated phosphofructokinase
gene and an introduced phosphoketolase gene, wherein a
phosphotransacetylase gene is introduced and/or the expression of an
acetyl-CoA synthetase gene is enhanced.

4. A recombinant yeast, which comprises an attenuated phosphofructokinase
gene and an introduced phosphoketolase gene, wherein the expression of an
alcohol acetyltransferase gene involved in a reaction for synthesis of
ethyl acetate using acetyl-CoA as a substrate is enhanced.

5. A recombinant yeast, which comprises an attenuated phosphofructokinase
gene and an introduced phosphoketolase gene, which is prepared by
introducing an acetoacetic acid decarboxylase gene, a
butyrate-acetoacetate CoA-transferase subunit A gene, a
butyrate-acetoacetate CoA-transferase subunit B gene, an acetyl-CoA
acetyltransferase gene, and an isopropanol dehydrogenase gene, which are
involved in a reaction for synthesis of isopropanol using acetyl-CoA as a
substrate.

6. A method for producing a substance, comprising a step of culturing the
recombinant yeast of claim 2 in medium.

7. The method for producing a substance according to claim 6, wherein the
substance is 1 type of substance selected from the group consisting of
acetic acid, acetyl-CoA, and a substance made from acetyl-CoA.

8. The method for producing a substance according to claim 7, wherein the
substance made from acetyl-CoA is ethyl acetate or isopropanol.

Description:

TECHNICAL FIELD

[0001] The present invention relates to a recombinant yeast prepared
through modification to suppress and/or enhance the expression of a
predetermined gene or introduction of a predetermined gene, and a
substance production method using the recombinant yeast.

BACKGROUND ART

[0002] Examples of techniques concerning substance production using yeast
are mainly methods for designing substance production pathways using
acetyl-CoA as an intermediate. For example, oleic acid, which is a
typical fatty acid, requires 9 molecules of acetyl-CoA as a raw material,
and carotin, which is a typical diterpene, requires 12 molecules of
acetyl-CoA as a raw material. Accordingly, a technique for synthesizing
fatty acid useful as a pharmaceutical product or a fine chemical (Patent
Document 1), a technique for synthesizing terpenoid (Patent Document 2),
and a technique for synthesizing polyketide (Patent Document 3) using
acetyl-CoA accumulated within yeast are known. Furthermore, examples of a
substance that is synthesized using acetyl-CoA as an intermediate include
butanol (Patent Document 4), isopropanol (Patent Document 5) and
farnesene (Patent Document 5), which are attracting attention as
biofuels.

[0003] In yeast, ethanol produced extracellularly is taken up by cells and
then acetyl-CoA is synthesized from the incorporated ethanol. When the
concentration of ethanol produced by yeast becomes high, the yeast's own
growth is inhibited. Therefore, it has been difficult to increase the
amount of acetyl-CoA within cells by means such as a means of increasing
the ethanol production capacity of yeast or a means of increasing the
amount of ethanol to be taken up by yeast.

[0004] More specifically, Patent Document 2 discloses a technique for
synthesizing farnesene from acetyl-CoA, but the yield thereof is about
25% of the theoretical yield. Moreover, Patent Document 6 discloses a
technique for synthesizing 6-methyl salicylate from acetyl-CoA, but the
yield is about 20% of the theoretical yield. As described above,
substance production from acetyl-CoA is problematic in that productivity
is significantly low. Prior Patent Documents

[0011] In view of the above circumstances, an object of the present
invention is to provide a recombinant yeast with high substance
productivity by introducing a metabolic pathway for synthesis of
acetyl-CoA or acetic acid from glucose-6-phosphate into yeast, in
particular, and a substance production method using the yeast.

Means for Solving Problem

[0012] As a result of intensive studies to achieve the above object, the
present inventors have discovered that the productivity of acetic acid,
acetyl-CoA, and a substance made from acetyl-CoA can be improved by
attenuating a gene involved in the glycolytic system of yeast and
introducing a phosphoketolase gene into the yeast, and thus they have
completed the present invention. In addition, the term "phosphoketolase"
refers to an enzyme that catalyzes a reaction to convert xylulose
5-phosphate into acetylphosphate and glyceraldehyde 3-phosphate.

[0013] Specifically, the present invention encompasses the following (1)
to (7).

[0014] (1) A recombinant yeast, which comprises an attenuated
phosphofructokinase gene and an introduced phosphoketolase gene.

[0015]
(2) The recombinant yeast according to (1), wherein the expression level
of a glucose-6-phosphate dehydrogenase gene and/or a
D-ribulose-5-phosphate-3-epimerase gene is increased.

[0016] (3) The
recombinant yeast according to (1), wherein a phosphotransacetylase gene
is introduced and/or the expression level of an acetyl-CoA synthetase
gene is increased.

[0017] (4) The recombinant yeast according to (1),
wherein the expression level of an alcohol acetyltransferase gene
involved in a reaction for synthesis of ethyl acetate using acetyl-CoA as
a substrate is increased.

[0018] (5) The recombinant yeast according to
(1), which is prepared by introducing an acetoacetic acid decarboxylase
gene, a butyrate-acetoacetate CoA-transferase subunit A gene, a
butyrate-acetoacetate CoA-transferase subunit B gene, an acetyl-CoA
acetyltransferase gene, and an isopropanol dehydrogenase gene, which are
involved in a reaction for synthesis of isopropanol using acetyl-CoA as a
substrate.

[0019] (6) A method for producing a substance, comprising a
step of culturing the recombinant yeast of any one of (1) to (5) above in
medium.

[0020] (7) The method for producing a material according to (6),
wherein the substance is 1 type of substance selected from the group
consisting of acetic acid, acetyl-CoA, ethyl acetate made from
acetyl-CoA, and isopropanol made from acetyl-CoA.

Effects of the Invention

[0021] The recombinant yeast according to the present invention has
attenuated activity of converting fructose-6-phosphate into
fructose-1,6-bisphosphate, and, imparted activity of converting xylulose
5-phosphate into acetylphosphate. Accordingly, through the use of the
recombinant yeast according to the present invention, the productivity of
acetic acid or a substance made from acetyl-CoA can be improved.

BRIEF DESCRIPTION OF THE DRAWINGS

[0022]FIG. 1 is a characteristic diagram showing a part of a glycolytic
system including a metabolic pathway in which a phosphofructokinase gene
to be attenuated in the yeast according to the present invention is
involved.

[0023]FIG. 2 is a characteristic diagram showing a part of a pentose
phosphate system including a metabolic pathway in which a phosphoketolase
gene to be introduced into the yeast according to the present invention
is involved.

[0024]FIG. 3 is a characteristic diagram showing the result of conducting
molecular phylogenetic tree analysis of phosphoketolase genes derived
from various organisms.

[0025]FIG. 4 is a characteristic diagram showing a pathway for synthesis
of acetyl-CoA from acetylphosphate and acetic acid and a pathway for
synthesis of another substance from acetyl-CoA.

[0026]FIG. 5 is a characteristic diagram showing the result of conducting
molecular phylogenetic tree analysis of phosphotransacetylase genes
derived from various organisms.

[0027]FIG. 6 is a flow chart for construction of a pESC-HIS-ZWF1-RPE1
vector.

[0028]FIG. 7 is a flow chart for construction of a pESC-Leu-PKT vector
and a pESC-Leu-PKT-PTA vector.

[0029]FIG. 8 is a flow chart for construction of a pESC-Trp-ATF1 vector.

[0030]FIG. 9 is a flow chart for construction of a vector for disruption
of a PFK1 gene.

[0031]FIG. 10 is a flow chart for construction of a vector for disruption
of a PFK2 gene.

[0032]FIG. 11 is a characteristic diagram showing the results of an
acetic acid production test.

[0033]FIG. 12 is a characteristic diagram showing the results of an ethyl
acetate production test.

[0034]FIG. 13 is a characteristic diagram showing the results of an
isopropanol production test.

[0035]FIG. 14 is a characteristic diagram showing the results of
conducting metabolome analysis of yeast by CE-TOFMS.

EMBODIMENTS FOR CARRYING OUT THE INVENTION

[0036] The present invention will be described in detail as follows with
reference to drawings and examples.

[0037] The recombinant yeast according to the present invention comprises
an attenuated gene that encodes an enzyme involved in a glycolytic
system, and an introduced phosphoketolase gene. The recombinant yeast has
activity to convert xylulose 5-phosphate to acetylphosphate. Examples of
yeast that can be used as a host include, but are not particularly
limited to, yeast of the genus Candida such as Candida Shehatae, yeast of
the genus Pichia such as Pichia stipitis, yeast of the genus Pachysolen
such as Pachysolen tannophilus, yeast of the genus Saccharomyces such as
Saccharomyces cerevisiae, and yeast of the genus Schizosaccharomyces such
as Schizosaccharomyces pombe. In particular, Saccharomyces cerevisiae is
preferred. A yeast strain to be used herein may be an experimental strain
to be used for convenience of experiments or an industrial strain
(practical strain) to be employed for practical usefulness. Examples of
such an industrial strain include yeast strains to be used for production
of wine, sake, and shochu (spirits).

[0038] Here, an example of a gene that encodes an enzyme involved in a
glycolytic system and is subjected to attenuation is a
phosphofructokinase gene.

[0039] Furthermore, as an enzyme involved in the glycolytic system,
hexokinase, glucose phosphate isomerase, aldolase, triosephosphate
isomerase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate
kinase, phosphoglyceromutase, enolase, and pyruvate kinase are known in
addition to phosphofructokinase. Genes encoding these enzymes other than
phosphofructokinase may also be attenuated.

[0040] The phosphofructokinase gene encodes an enzyme that converts
fructose-6-phosphate into fructose-1,6-bisphosphate in the glycolytic
system, as shown in FIG. 1. The expression, "the phosphofructokinase gene
is attenuated" means that phosphofructokinase activity is significantly
lowered. In other words, the expression means that the amount of
fructose-1,6-bisphosphate to be synthesized through the glycolytic system
is significantly decreased. Examples of means for attenuating a
phosphofructokinase gene include, but are not particularly limited to,
disruption or deletion of the phosphofructokinase gene, disruption or
deletion of the expression control region of the phosphofructokinase
gene, addition of an inhibitor (e.g., citric acid) of
phosphofructokinase, and a technique for suppressing the expression of
the phosphofructokinase gene with the use of a method using RNA
interference such as siRNA or an antisense method.

[0041] In addition, as endogenous phosphofructokinase genes of
Saccharomyces cerevisiae, a PFK1 gene and a PFK2 gene are known (THE
JOURNAL OF BIOLOGICAL CHEMISTRY, Vol. 275, No. 52, Issue of December 29,
pp. 40952-40960, 2000). When Saccharomyces cerevisiae is used as a host
for the recombinant yeast according to the present invention, either the
PFK1 gene or the PFK2 gene may be attenuated or both genes may be
attenuated. Moreover, endogenous phosphofructokinase genes of yeast other
than Saccharomyces cerevisiae are also known and can be specified
referring to existing databases such as Genbank, DDBJ, and EMBL. As
described above, phosphofructokinase genes specified by the above
techniques and/or means can be attenuated by specifying endogenous
phosphofructokinase genes of various types of yeast.

[0042] Furthermore, the recombinant yeast according to the present
invention acquires the capacity to convert xylulose 5-phosphate into
acetylphosphate through exogenous introduction of a phosphoketolase gene
(PKT gene). In addition, xylulose 5-phosphate is synthesized as a
metabolite resulting from yeast's original pentose phosphate system from
ribulose-5-phosphate (FIG. 2). Acetylphosphate synthesized by
phosphoketolase is converted into acetic acid by yeast's original acetic
acid kinase. Therefore, in the recombinant yeast prepared by attenuating
a phosphofructokinase gene and introducing a phosphoketolase gene, the
productivity of acetic acid to be secreted to medium is drastically
improved.

[0043] Examples of phosphoketolase genes to be preferably used herein
include, but are not particularly limited to, phosphoketolase genes
derived from lactic acid bacteria or bifidobacteria having a metabolic
pathway for heterolactic fermentation. Here, the term "heterolactic
fermentation" refers to fermentation whereby pyruvic acid generated via
the glycolytic system from glucose is metabolized to give not only lactic
acid, but also ethanol, acetic acid, and carbon dioxide. Through such
heterolactic fermentation, ethanol or acetic acid is synthesized from
acetylphosphate that is generated by phosphoketolase.

[0044] More specifically, as phosphoketolase genes, as shown in FIG. 3,
phosphoketolase genes derived from various microorganisms can be used. In
addition, Table 1 below shows the relationship between numbers assigned
in the molecular phylogenetic tree shown in FIG. 3 and microorganisms.

[0047] Furthermore, a phosphoketolase gene may consist of a polynucleotide
encoding a protein that consists of an amino acid sequence having a
deletion, a substitution, an addition, or an insertion of 1 or several
amino acids with respect to the amino acid sequence of any one of SEQ ID
NOS: 1 to 19 and has phosphoketolase activity. Here, the term "several
amino acids" refers to, for example, 2 to 100, preferably 2 to 80, more
preferably 2 to 55, and further preferably 2 to 15 amino acids.

[0048] Furthermore, a phosphoketolase gene may consist of a polynucleotide
encoding a protein that consists of an amino acid sequence having 80% or
more, preferably 85% or more, more preferably 90% or more, and further
preferably 98% or more sequence similarity with respect to the amino acid
sequence of any one of SEQ ID NOS: 1 to 19, and has phosphoketolase
activity. Here, the term "sequence similarity" refers to a value that is
calculated to represent similarity between two amino acid sequences when
sequence similarity search software such as BLAST, PSI-BLAST, or HMMER is
used with default settings.

[0049] Here, the term "phosphoketolase activity" refers to activity to
convert xylulose-5-phosphate to acetylphosphate. Therefore, whether or
not a predetermined protein has phosphoketolase activity can be
determined based on the amount of acetylphosphate synthesized, using a
reaction solution containing xylulose-5-phosphate as a substrate (e.g.,
JOURNAL OF BACTERIOLOGY, Vol. 183, No. 9, May 2001, p. 2929-2936).

[0050] The recombinant yeast according to the present invention can
increase the amount of acetylphosphate synthesized because of the
presence of a phosphoketolase gene introduced, by enhancing the
expression of an enzyme gene involved in the pentose phosphate system
shown in FIG. 2. As a result, it can increase the amount of synthesized
acetic acid. Examples of genes to be subjected to enhancement of
expression in the pentose phosphate system shown in FIG. 2 include, but
are not particularly limited to, a glucose-6-phosphate dehydrogenase gene
and a ribulose-5-phosphate-3-epimerase gene. Moreover, the expression of
either one of or both of these genes may be enhanced.

[0051] Furthermore, an endogenous glucose-6-phosphate dehydrogenase gene
of Saccharomyces cerevisiae is known as a ZWF1 gene. Also, an endogenous
ribulose-5-phosphate-3-epimerase gene of Saccharomyces cerevisiae is
known as an RPE1 gene. Endogenous glucose-6-phosphate dehydrogenase genes
or ribulose-5-phosphate-3-epimerase genes are known for yeast other than
Saccharomyces cerevisiae and can be specified referring to the existing
databases such as Genbank, DDBJ, and EMBL.

[0052] Here, the expression "gene expression is enhanced" refers to
significant improvement in activity of an enzyme to be encoded by a
subject gene, and is meant to include a significant increase in the
expression level of such a gene. An example of a technique for enhancing
gene expression is a technique for significantly increasing the
expression level of the relevant gene. Examples of a technique for
increasing the expression level of a specific gene include, but are not
particularly limited to, a technique that involves modifying the
expression control region of an endogenous gene of a chromosome and a
technique that involves introducing a vector having the relevant gene
located downstream of a promoter with high activity.

[0053] Meanwhile, the recombinant yeast according to the present invention
can increase the amount of acetyl-CoA synthesized by further introducing
a phosphotransacetylase gene (PTA gene) or further enhancing an
acetyl-CoA synthetase gene (ACS gene) as shown in FIG. 4, in addition to
attenuation of a phosphofructokinase gene and introduction of a
phosphoketolase gene.

[0054] A phosphotransacetylase gene is not yeast's original gene and thus
is introduced as a foreign gene. Examples of such a phosphotransacetylase
gene are not particularly limited, and genes referred to as PTA genes in
various bacteria are broadly applicable.

[0055] More specifically, as phosphotransacetylase genes, as shown in FIG.
5, phosphotransacetylase genes derived from various microorganisms can be
used. In addition, Table 2 below shows the relationship between numbers
assigned in the molecular phylogenetic tree in FIG. 5 and microorganisms.

[0056] The origins of the following PTA genes are shown in FIG. 5 and
Table 2. The amino acid sequence of a protein encoded by the Bacillus
subtilis-derived PTA gene is shown in SEQ ID NO: 20, the amino acid
sequence of a protein encoded by the Bacillus amyloliquefaciens-derived
PTA gene is shown in SEQ ID NO: 21, the amino acid sequence of a protein
encoded by the Bacillus licheniformis-derived PTA gene is shown in SEQ ID
NO: 22, the amino acid sequence of a protein encoded by the Geobacillus
thermodenitrificans-derived PTA gene is shown in SEQ ID NO: 23, the amino
acid sequence of a protein encoded by the Listeria innocua-derived PTA
gene is shown in SEQ ID NO: 24, the amino acid sequence of a protein
encoded by the Staphylococcus aureus-derived PTA gene is shown in SEQ ID
NO: 25, the amino acid sequence of a protein encoded by the Lactococcus
lactis-derived PTA gene is shown in SEQ ID NO: 26, the amino acid
sequence of a protein encoded by the Carnobacterium sp.-derived PTA gene
is shown in SEQ ID NO: 27, the amino acid sequence of a protein encoded
by the Mycobacterium vanbaalenii-derived PTA gene is shown in SEQ ID NO:
28, the amino acid sequence of a protein encoded by the Clostridium
perfringens-derived PTA gene is shown in SEQ ID NO: 29, the amino acid
sequence of a protein encoded by the Enterococcus faecalis-derived PTA
gene is shown in SEQ ID NO: 30, the amino acid sequence of a protein
encoded by the Leuconostoc mesenteroides-derived PTA gene is shown in SEQ
ID NO: 31, the amino acid sequence of a protein encoded by the
Clostridium acetobutylicum-derived PTA gene is shown in SEQ ID NO: 32,
the amino acid sequence of a protein encoded by the Bifidobacterium
animalis--lactis-derived PTA gene is shown in SEQ ID NO: 33, the
amino acid sequence of a protein encoded by the Corynebacterium
glutamicum-derived PTA gene is shown in SEQ ID NO: 34, the amino acid
sequence of a protein encoded by the Escherichia coli K-12-derived PTA
gene is shown in SEQ ID NO: 35, the amino acid sequence of a protein
encoded by the Escherichia coli 53638-derived PTA gene is shown in SEQ ID
NO: 36, the amino acid sequence of a protein encoded by the Vibrio
vulnificus-derived PTA gene is shown in SEQ ID NO: 37, the amino acid
sequence of a protein encoded by the Haemophilus somnus-derived PTA gene
is shown in SEQ ID NO: 38, the amino acid sequence of a protein encoded
by the Yersinia pestis-derived PTA gene is shown in SEQ ID NO: 39, and
the amino acid sequence of a protein encoded by the Shigella
sonnei-derived PTA gene is shown in SEQ ID NO: 40.

[0057] In addition, the phosphotransacetylase gene may consist of a
polynucleotide encoding a protein that consists of an amino acid sequence
having a deletion, a substitution, an addition, or an insertion of 1 or
several amino acids with respect to the amino acid sequence of any one of
SEQ ID NOS: 20 to 40, and has phosphotransacetylase activity. Here, the
term "several amino acids" refers to, for example, 2 to 35, preferably 2
to 25, more preferably 2 to 15, and further preferably 2 to 10 amino
acids.

[0058] Furthermore, the phosphotransacetylase gene may consist of a
polynucleotide encoding a protein that consists of an amino acid sequence
having 80% or more, preferably 85% or more, more preferably 90% or more,
and further preferably 98% or more sequence similarity with respect to
the amino acid sequence of any one of SEQ ID NOS: 20 to 40, and has
phosphotransacetylase activity. Here, the term "sequence similarity"
refers to a value that is calculated to represent similarity between two
amino acid sequences when sequence similarity search software such as
BLAST, PSI-BLAST, or HMMER is used with default settings.

[0059] Here, the term "phosphotransacetylase activity" refers to activity
to transfer CoA to acetylphosphate. Therefore, whether or not a
predetermined protein has phosphotransacetylase activity can be
determined based on the amount of acetyl-CoA synthesized using a reaction
solution containing acetylphosphate and CoA.

[0060] Furthermore, the acetyl-CoA synthetase gene shown in FIG. 4 is
yeast's original gene. Hence, for enhancement of such an acetyl-CoA
synthetase gene, a technique that involves modifying the expression
control region of the relevant endogenous gene of a chromosome and a
technique that involves introducing a vector having the relevant gene
located downstream of a promoter having high activity are applicable. In
addition, as endogenous acetyl-CoA synthetase genes of Saccharomyces
cerevisiae, an ACS1 gene and an ACS2 gene are known. Endogenous
acetyl-CoA synthetase genes of yeast other than Saccharomyces cerevisiae
are also known and can be specified referring to existing databases such
as Genbank, DDBJ, and EMBL.

[0061] As described above, in the recombinant yeast according to the
present invention, the amount of acetylphosphate synthesized; that is,
the amount of acetic acid synthesized is significantly increased (FIG. 2)
or the amount of acetyl-CoA synthesized is significantly increased (FIG.
4). Therefore, the recombinant yeast according to the present invention
can be used when acetic acid or acetyl-CoA is a substance to be produced.
Alternatively, the recombinant microorganism according to the present
invention can be used as a host for further modification to enable
production of another substance (in FIG. 4, denoted as a substance made
from acetyl-CoA) using acetyl-CoA as a substrate.

[0062] Specifically, examples of such a substance made from acetyl-CoA
that can be synthesized include, but are not particularly limited to,
butanol, alkane, propanol, fatty acid, fatty acid ester, acetone,
acetoacetic acid, ethyl acetate, polyketide, amino acid, and terpenoid.
When these are substances to be produced, the productivity thereof can be
significantly improved using the recombinant yeast according to the
present invention.

[0063] When isopropanol is a substance to be produced, for example, with
reference to APPLIED AND ENVIRONMENTAL MICROBIOLOGY, December 2007, p.
7814-7818, Vol. 73, No. 24, a gene to be further introduced into the
recombinant microorganism according to the present invention can be
specified. Furthermore, when polyketide is a substance to be produced,
with reference to Proc. Natl. Acad. Sci. U.S.A., Vol. 95, pp. 505-509,
January 1998, a gene to be further introduced into the recombinant
microorganism according to the present invention can be specified.
Moreover, when fatty acid is a substance to be produced, for example,
with reference to Eur. J. Biochem. 112, p. 431-442 (1980) or MICROBIOLOGY
AND MOLECULAR BIOLOGY REVIEWS, September 2004, p. 501-517, a gene (e.g.,
a FAS gene) to be further introduced to or enhanced in the recombinant
microorganism according to the present invention can be specified.
Furthermore, when alkane is a substance to be produced, a gene that is
involved in aldehyde synthesis from fatty acid and further alkane
synthesis from aldehyde and should be further introduced into the
recombinant microorganism according to the present invention can be
specified with reference to Science vol. 329 30 July pp. 559-562, for
example.

[0064] Furthermore, the expression of an endogenous alcohol
acetyltransferase gene (ATF1 gene) of yeast is further enhanced, so that
the amount of ethyl acetate synthesized from acetyl-CoA can be increased.
Specifically, when ethyl acetate is a substance to be produced, the
expression of such an endogenous ATF1 gene is preferably enhanced.

[0065] Also, as described above, when the expression of a predetermined
gene is enhanced, an appropriate promoter with high transcriptional
activity is used. Examples of such a promoter that can be used herein
include, but are not particularly limited to, a
glyceraldehyde-3-phosphate dehydrogenase gene (TDH3) promoter, a
3-phosphoglyceratekinase gene (PGK1) promoter, and a high osmotic
pressure-responsive 7 gene (HOR7) promoter. Of these, a pyruvate
decarboxylase gene (PDC1) promoter is preferred because of its high
capacity to cause high-level expression of a gene of interest located
downstream thereof. Furthermore, through the use of a gall promoter, a
gal10 promoter, a heat shock protein promoter, a MFα1 promoter, a
PHO5 promoter, a GAP promoter, an ADH promoter, an AOX1 promoter, or the
like, a gene downstream thereof can be strongly expressed.

[0066] Also, as a method for introducing the above gene, any
conventionally known technique that is known as a method for yeast
transformation is applicable. Specifically, for example, gene
introduction can be performed by a method described in an electroporation
method "Meth. Enzym., 194, p182 (1990)," a spheroplast method "Proc.
Natl. Acad. Sci. U.S.A., 75 p1929 (1978)," a lithium acetate method "J.
Bacteriology, 153, p 163 (1983)," Proc. Natl. Acad. Sci. U.S.A., 75 p
1929 (1978), Methods in yeast genetics, 2000 Edition: A Cold Spring
Harbor Laboratory Course Manual, or the like. Examples thereof are not
limited to these methods.

[0067] When a substance is produced using the above-explained recombinant
yeast, the yeast is cultured in a medium containing an appropriate carbon
source. More specifically, recombinant yeast pre-cultured according to a
conventional method is cultured in a medium so as to cause it to produce
a substance of interest. For example, when butanol, alkane, propanol,
fatty acid, fatty acid ester, acetone, acetoacetic acid, acetic ester,
polyketide, amino acid, and terpenoid are produced as substances of
interest, these substances of interest are synthesized in a medium.
Hence, after cells are separated from the medium by a means such as
centrifugation, such substances can be isolated from the supernatant
fraction. To isolate such substances from a supernatant fraction, for
example, an organic solvent such as ethyl acetate or methanol is added to
the supernatant fraction, and then the resultant is sufficiently stirred.
An aqueous layer is separated from a solvent later, and then the
substances can be extracted from the solvent layer.

EXAMPLES

[0068] The present invention is hereafter described in greater detail with
reference to the following examples, although the technical scope of the
present invention is not limited thereto.

[0069] In these examples, recombinant yeast was prepared by attenuating
the endogenous phosphofructokinase gene of wild-type yeast or
isopropanol-producing yeast and introducing a phosphoketolase gene into
the yeast. Recombinant yeast was further prepared by introducing or
enhancing other genes in addition to the aforementioned gene attenuation
and gene introduction. These strains were then examined for acetic acid
productivity, ethyl acetate productivity, and isopropanol productivity.

[0078] After PCR under the above conditions, a PCR product contained in
the reaction solution was purified using a MinElute PCR purification kit
(QIAGEN). Subsequently, the PCR product was digested with restriction
enzymes Barn HI and EcoR I. Agarose gel electrophoresis was performed, a
686-bp fragment was excised, and the fragment was thus purified using a
MiniElute Gel extraction kit (QIAGEN). Furthermore, the resultant was
ligated to a pESC-HIS vector digested with restriction enzymes Barn HI
and EcoR I. The thus obtained plasmid was designated as pESCgap-HIS.

[0085] After PCR under the above conditions, a PCR product contained in
the reaction solution was purified using a MinElute PCR purification kit
(QIAGEN). Subsequently, the PCR product was digested with restriction
enzymes Mun I and EcoR I. Agarose gel electrophoresis was performed, a
718-bp fragment was excised, and the fragment was thus purified using a
MiniElute Gel extraction kit (QIAGEN). Furthermore, the resultant was
ligated to a pESCgap-HIS vector digested with a restriction enzyme EcoR I
and then subjected to BAP treatment. The thus obtained plasmid was
designated as pESCpgkgap-HIS.

<Construction of pESCpgkgap-LEU>

[0086] After digestion of the above pESCpgkgap-HIS with restriction
enzymes Barn HI and Not I, a 1427-bp fragment was excised and ligated to
a pESC-LEU vector (Stratagene) digested with restriction enzymes Barn HI
and Not I in a similar manner.

<Construction of pESCpgkgap-TRP>

[0087] After digestion of the above pESCpgkgap-HIS with restriction
enzymes Barn HI and Not I, a 1427-bp fragment was excised and then
ligated to a pESC-TRP vector (Stratagene) digested with restriction
enzymes Barn HI and Not I in a similar manner.

<Construction of pESCpgkgap-URA>

[0088] After digestion of the above pESCpgkgap-HIS with restriction
enzymes Barn HI and Not I, a 1427-bp fragment was excised and then
ligated to a pESC-URA vector (Stratagene) digested with restriction
enzymes Barn HI and Not I in a similar manner.

<Construction of Other Vectors>

[0089] Upon construction of a pESC-HIS-ZWF1-RPE1 vector for enhancing the
expression of a ZWF1 gene and a RPE1 gene, a pESC-Leu-PKT vector for
introducing a PKT gene, a pESC-Leu-PKT-PTA vector for introducing the PKT
gene and a PTA gene, a pESC-Trp-ATF1 vector for enhancing the expression
of an ATF1 gene, and a vector for disrupting a PFK1 gene and a PFK2 gene,
PCR was performed with the following composition under the following
conditions. In addition, KOD-Plus-Ver.2 (TOYOBO) was used as DNA
polymerase.

[0090]FIG. 6 shows a flow chart for construction of the
pESC-HIS-ZWF1-RPE1 vector. FIG. 7 shows a flow chart for construction of
the pESC-Leu-PKT vector and the pESC-Leu-PKT-PTA vector. FIG. 8 shows a
flow chart for construction of the pESC-Trp-ATF1 vector. FIG. 9 shows a
flow chart for construction of the vector for disrupting the PFK1 gene.
FIG. 10 shows a flow chart for construction of the vector for disrupting
the PFK2 gene. Primers used in the flow charts for vector construction
shown in FIGS. 6-10 are listed in Table 5.

[0091] In addition, PCR products were purified using a QIAquick PCR
Purification Kit (QIAGEN). Furthermore, in the flow charts for vector
construction as shown in FIGS. 6 to 10, a Zero Blunt TOPO PCR cloning kit
(Invitrogen) was used for TOPO cloning of PCR products. Also, pAUR135 was
purchased from Takara Bio Inc. In the flow charts for vector construction
as shown in FIGS. 6 to 10, DNA fragments were excised from agarose gel
using a QIAquick Gel Extraction Kit (QIAGEN). In the flow charts for
vector construction as shown in FIGS. 6 to 10, vectors were ligated to
DNA fragments by ligation reaction using a Ligation-convenience Kit
(Nippon Gene Co., Ltd.) or in-fusion reaction using an In-Fusion Dry-Down
PCR Cloning Kit (Clontech).

[0092] Furthermore, in the flow chart for vector construction as shown in
FIG. 7, vectors denoted as pBSK-PKT and pBSK-PTA were constructed by
synthesizing 5 types of PKT gene and 3 types of PTA gene, respectively,
and then inserting them to the Sma I site of pBluescript IISK(+). In
addition, these 5 types of PKT gene and 3 types of PTA gene were all
optimized for codons for Saccharomyces cerevisiae.

[0094] Escherichia coli was transformed according to the protocols
included with ECOS Competent E. coli JM109 (Nippon Gene Co., Ltd.). Yeast
was transformed according to the protocols included with a Frozen-EZ
Yeast Transformation II Kit (Zymo Research). Yeast gene disruption using
the vector for disrupting the PFK1 gene and the PFK2 gene was performed
according to the protocols included with pAUR135DNA (Takara Bio Inc.).

Acetic Acid Production Test

[0095] A transformant was cultured as follows. After active colony
formation in an SD-His, Leu agar medium, cells were inoculated to 2 ml of
an SD-His, Leu medium in a 15-ml test tube and then cultured overnight at
30 degrees C. (Oriental Giken Inc. IFM, 130 rpm). The thus pre-cultured
solution was inoculated to 100 ml of an SD-His, Leu medium in a 500-ml
Erlenmeyer flask so that it accounted for 1% of the volume of the medium,
and then cultured. The culture solution was centrifuged (6000×g, 15
min, room temperature) and then 1 ml of the supernatant was introduced
into a glass vial and thus designated as an analysis sample.

Ethyl Acetate Production Test

[0096] A transformant was cultured as follows. After active colony
formation in an SD-His, Leu, Trp agar medium, cells were inoculated to 2
ml of an SD-His, Leu, Trp medium in a 15-ml test tube and then cultured
overnight at 30 degrees C. (130 rpm). The thus pre-cultured solution was
inoculated to 100 ml of an SD-His, Leu, Trp medium in a 500-ml Erlenmeyer
flask so that it accounted for 1% thereof, and then cultured. The culture
solution was centrifuged (6000×g, 15 min, room temperature) and
then 1 ml of the supernatant was introduced into a glass vial so that it
was designated as an analysis sample.

Isopropanol Production Test

[0097] A transformant was cultured as follows. After active colony
formation in an SD-His, Leu, Ura, Trp agar medium, cells were inoculated
to 2 ml of an SD-His, Leu, Ura, Trp medium in a 15-ml test tube and then
cultured overnight at 30 degrees C. (130 rpm). The thus pre-cultured
solution was inoculated to 50 ml of an SD-His, Leu, Ura, Trp medium in a
500-ml Erlenmeyer flask so that it accounted for 1% thereof, and then
cultured. The culture solution was centrifuged (6000×g, 15 min,
room temperature) and then 1 ml of the supernatant was introduced into a
glass vial so that it was designated as an analysis sample.

GC Analysis Conditions

[0098] Preparations used herein were acetic acid (NACALAI TESQUE, INC.),
ethyl acetate (NACALAI TESQUE, INC.), and isopropanol (NACALAI TESQUE,
INC.). The following analytical instrument and analysis conditions were
employed for the acetic acid production test, the ethyl acetate
production test and the isopropanol production test.

[0099] Cells were cultured (30 degrees C.) in an SD-His, Leu, Ura, Trp
medium and then sampling was performed so that the amount of the sample
was 15 OD unit. Suction filtration of the resultant was immediately
performed by filtration. Next, suction filtration was performed twice
with 10 mL of Milli-Q water and then washing was performed. Yeast cells
collected on the filter were immersed in 2 mL of methanol containing
internal reference material (5 μM) and then 1.6 mL of the resultant
was transferred into a centrifugation tube. 1600 μL of chloroform and
640 μL of Milli-Q water were added to the tube and then it was
stirred, followed by centrifugation (2,300×g, 4 degrees C., 5
minutes). After centrifugation, aqueous phase was transferred to 6
ultrafiltration tubes (250 μL each) (MILLIPORE, Ultrafree MC UFC3 LCC
Centrifugal Filter Unit 5 KDa). The tubes were centrifuged
(9,100×g, 4 degrees C., 120 minutes) and thus ultrafiltration was
performed. Each filtered fluid was solidified to dryness, dissolved again
in 50 μL of Milli-Q water, and then subjected to measurement.

<Measurement>

[0100] In this test, anionic metabolite (anion mode) measurement was
performed under the following conditions (see references 1) to 3)).

[0118] Based on the results shown in FIG. 11, it was revealed that
attenuation of an endogenous phosphofructokinase gene of yeast as a host
and introduction of a phosphoketolase gene into the yeast can enhance the
flux toward the pentose phosphate system rather than toward the
glycolytic system (FIG. 2), so that acetic acid can be produced at a high
level. It was also revealed that as a phosphoketolase gene, the
Bifidobacterium-derived gene, and particularly the Bifidobacterium
animalis-derived gene, is excellent in acetic acid productivity. It was
revealed based on the results that as phosphoketolase genes,
phosphoketolase genes within the broken-line frame in the phylogenetic
tree shown in FIG. 3 are preferable in terms of acetic acid productivity.

[0119] Furthermore, it was revealed based on the results shown in FIG. 11
that when a phosphofructokinase gene is attenuated to weaken the flux
toward the glycolytic system, both the PFK1 gene and the PFK2 gene are
preferably disrupted. Moreover, it was revealed that through enhancement
of enzyme genes involved in the pentose phosphate system (FIG. 2), the
flux toward the pentose phosphate system (FIG. 2) can be further enhanced
and acetic acid can be produced at an even higher level.

Ethyl Acetate Production Test

[0120] Yeast strains subjected to the ethyl acetate production test are
listed in Table 8 and the test results are shown in FIG. 12.

[0122] As shown in FIG. 12, the control strain in which the ATF1 gene had
been introduced into the wild-type strain (YPH499 strain) also exhibited
more improved ethyl acetate productivity compared with that of the
wild-type strain. However, it was understood that through attenuation of
an endogenous phosphofructokinase gene of yeast as a host and
introduction of a phosphoketolase gene into the host, in addition to
further introduction of a phosphoacetyltransferase gene (PTA gene) or
enhancement of an acetyl-CoA synthetase gene (ACS gene), ethyl acetate
productivity was further improved compared with that of the control. It
was demonstrated based on the results and the metabolic overview map
shown in FIG. 4 that the amount of acetyl-CoA synthesized was
significantly increased through attenuation of the endogenous
phosphofructokinase gene and introduction of the phosphoketolase gene in
addition to further introduction of the phosphoacetyltransferase gene
(PTA gene) or enhancement of the acetyl-CoA synthetase gene (ACS gene).
Moreover, it was demonstrated that the thus synthesized acetyl-CoA is
accumulated extracellularly at a high level in the form of ethyl acetate
by an ATF1 enzyme.

[0123] In particular, as a phosphoacetyltransferase gene to be introduced,
a Bacillus subtilis-derived gene is preferable in view of acetyl-CoA
productivity. Also, it was revealed that as an acetyl-CoA synthetase gene
to be enhanced, the ACS1 gene is more preferable than the ACS2 gene.

[0124] Furthermore, it was revealed based on the results shown in FIG. 12
that when a phosphofructokinase gene is attenuated to weaken the flux
toward the glycolytic system, both the PFK1 gene and the PFK2 gene are
preferably disrupted. Moreover, it was revealed that through enhancement
of enzyme genes involved in the pentose phosphate system (FIG. 2), the
flux toward the pentose phosphate system (FIG. 2) can be increased more
and ethyl acetate can be produced at an even higher level.

Isopropanol Production Test

[0125] Yeast strains subjected to the isopropanol production test are
listed in Table 9 and the test results are shown in FIG. 13.

[0126] In Table 9, PTA genes (1) to (3) denote the same genes as in Table
8.

[0127] As shown in FIG. 13, it was understood that the capacity to produce
isopropanol can be imparted to yeast through introduction of a ctfA gene,
a ctfB gene, an adc gene, and an ipdh gene into the wild-type strain
(YPH499 strain). It was also understood that isopropanol productivity was
significantly improved compared with that of the control through
attenuation of the endogenous phosphofructokinase gene and introduction
of the phosphoketolase gene, in addition to further introduction of the
phosphoacetyltransferase gene (PTA gene) or enhancement of the acetyl-CoA
synthetase gene (ACS gene).

[0128] It was demonstrated based on the results and the metabolic overview
map shown in FIG. 4 that the amount of acetyl-CoA synthesized was
significantly increased through attenuation of the endogenous
phosphofructokinase gene and introduction of the phosphoketolase gene, in
addition to further introduction of the phosphoacetyltransferase gene
(PTA gene) or enhancement of the acetyl-CoA synthetase gene (ACS gene).
It was demonstrated that the thus synthesized acetyl-CoA is accumulated
extracellularly at a high level as isopropanol because of ctfA, ctfB,
adc, and ipdh.

[0129] In particular, it was understood that as a phosphoacetyltransferase
gene to be introduced, Bacillus subtilis-derived gene is preferable in
view of acetyl-CoA productivity. Furthermore, it was revealed that as an
acetyl-CoA synthetase gene to be enhanced, the ACS1 gene is more
preferable than the ACS2 gene.

Metabolome Analysis of Yeast by CE-TOFMS

[0130] Metabolome analysis was conducted for: a wild-type strain (YPH499
strain); a strain (denoted as YPH499ΔPFKα/ZWF-RPE) prepared
by attenuating an endogenous phosphofructokinase gene of the YPH499
strain and enhancing an enzyme gene involved in the pentose phosphate
system (FIG. 2); and a strain (denoted as
YPH499ΔPFKα/ZWF-RPE, PKT-PTA) prepared by attenuating the
endogenous phosphofructokinase gene of the YPH499 strain, enhancing an
enzyme gene involved in the pentose phosphate system (FIG. 2), and
introducing a phosphoketolase gene and a phosphoacetyltransferase gene.
FIG. 14 shows the results of the metabolome analysis.

[0131] In FIG. 14, the amount of acetyl-CoA in the case of the YPH499
strain (wild-type strain) is found by adding up the amount resulting from
the ethanol-mediated acetyl-CoA synthetic pathway and the amount
resulting from the intramitochondrial acetyl-CoA synthetic pathway. In
addition, in yeast's acetyl-CoA synthetic pathway, these two pathways,
the ethanol-mediated acetyl-CoA synthetic pathway and the
intramitochondrial acetyl-CoA synthetic pathway, are present.

[0132] Meanwhile, in the case of the YPH499ΔPFKα/ZWF-RPE
strain, the ethanol-mediated acetyl-CoA synthetic pathway becomes
unfunctional. This is because NADP is consumed because of the presence of
ZWF, and as a result, ALD2 catalyzing the reaction for conversion of
aldehyde to acetic acid becomes unfunctional (see acetic acid
productivity of the control strain in the acetic acid production test
(FIG. 11)). Therefore, the amount of acetyl-CoA synthesized by the
YPH499ΔPFKα/ZWF-RPE strain corresponds to the amount of
acetyl-CoA generated via the intramitochondrial acetyl-CoA synthetic
pathway. In addition, intramitochondrially synthesized acetyl-CoA is
consumed within mitochondria, and thus it cannot be used as a raw
material for substances such as ethyl acetate and isopropanol. Therefore,
unless the amount of acetyl-CoA to be synthesized in the cytosol of yeast
cells is increased, the amounts of substances to be synthesized using
synthesized acetyl-CoA cannot be increased.

[0133] The YPH499ΔPFKα/ZWF-RPE, PKT-PTA strain is a mutant
strain prepared by introducing the PKT gene and the PTA gene into the
YPH499ΔPFKα/ZWF-RPE strain, so as to construct a novel
acetyl-CoA synthetic pathway (see FIG. 2 and FIG. 4). Therefore, the
amount of acetyl-CoA synthesized via the above novel acetyl-CoA synthetic
pathway can be evaluated by subtracting the amount of acetyl-CoA
synthesized by the YPH499ΔPFKα/ZWF-RPE strain from the amount
of acetyl-CoA synthesized by the YPH499ΔPFKα/ZWF-RPE, PKT-PTA
strain. Specifically, it was concluded from the results shown in FIG. 14
that the amount of acetyl-CoA corresponding to 48 pmol (found by 62-14=48
pmol) could be synthesized via the novel acetyl-CoA synthetic pathway.

REFERENCE EXAMPLE

[0134] In the reference example, preparation of the isopropanol-producing
yeast (#15-10) used in the above examples is described.

Obtainment of Isopropanol Synthetic Gene

[0135] The following 4 genes were cloned from the genome of Clostridium
acetobutylicum ATCC824 strain to a pT7Blue vector.

[0146] The thus amplified 735-bp fragment was blunt-end cloned to a
pT7Blue vector using a Perfectly Blunt Cloning Kit (Novagen). The cloned
sequence was sequenced, thereby confirming that it was the adc sequence
(CA-P0165) of the Clostridium acetobutylicum ATCC824 strain. The thus
obtained plasmid was designated as pT7Blue-ADC.

[0153] The thus amplified 657-bp fragment was cloned to a Perfectly Blunt
Cloning Kit (Novagen) in a similar manner. The cloned sequence was
sequenced, thereby confirming that it was the ctfA sequence (CA-P0163) of
the Clostridium acetobutylicum ATCC824 strain. The thus obtained plasmid
was designated as pT7Blue-CTFA.

[0160] The thus amplified 666-bp fragment was cloned using a Perfectly
Blunt Cloning Kit (Novagen). The cloned sequence was sequenced, thereby
confirming that it was the ctfB sequence (CA-P0164) of the Clostridium
acetobutylicum ATCC824 strain. The thus obtained plasmid was designated
as pT7Blue-CTFB.

[0167] After purification of the reaction solution using a MinElute PCR
purification kit (QIAGEN), the resultant was digested with restriction
enzymes Sac I and Sac II. Agarose gel electrophoresis was performed to
excise a 712-bp fragment, and then it was purified using a MinElute Gel
extraction kit (QIAGEN). The resultant was ligated to a pDI626GAP (APP.
Env. Micro., 2009, 5536) vector digested with restriction enzymes Sac I
and Sac II in a similar manner. The thus obtained sequence was seqeunced,
thereby confirming that a plasmid of interest had been constructed. The
thus obtained plasmid was designated as pDI626PGKpro.

[0174] After purification of the reaction solution using a MinElute PCR
purification kit (QIAGEN), the resultant was digested with restriction
enzymes Sal I and Kpn I. Agarose gel electrophoresis was performed to
excise a 330-bp fragment, and then it was purified using a MinElute Gel
extraction kit (QIAGEN). The resultant was ligated to a pDI626PGKpro
vector digested with restriction enzymes Sal I and Kpn I. The thus
obtained sequence was sequenced, thereby confirming that a plasmid of
interest had been constructed. The thus obtained plasmid was designated
as pDI626PGK.

Construction of pDI626PGK-T

[0175] pDI626PGK was digested with a restriction enzyme Sbf I, and then
the reaction solution was purified using a MinElute PCR purification kit
(QIAGEN). Subsequently, the resultant was blunt-ended using a Blunting
kit (TaKaRaBIO), and then further digested with a restriction enzyme Kpn
I. Agarose gel electrophoresis was performed to excise a 3650-bp
fragment, and then it was purified using a MinElute Gel extraction kit
(QIAGEN). Thus a vector for ligation thereof was constructed. Next,
pRS524GAP (APP. Env. Micro., 2009, 5536) was digested with restriction
enzymes PmaC I and Kpn I. Agarose gel electrophoresis was performed to
excise a 765-bp fragment and then it was purified using a MinElute Gel
extraction kit (QIAGEN), so as to prepare an insert. Ligation thereof was
performed. Joints of the thus obtained sequence were sequenced, thereby
confirming that a plasmid of interest had been constructed. The thus
obtained plasmid was designated as pDI626PGK-T.

Construction of pCR2.1-iPDH

[0176] A DNA sequence optimized for Saccharomyces cerevisiae codons based
on the Clostridium beijerinckii NRRL B593-derived adh: NADP-dependent
alcohol dehydrogenase gene sequence registered in the GenBank
(http://www.neb.nih.gov/Genbank/index.html) was synthesized (Operon). A
vector portion is pCR2.1 (Invitrogen). In addition, the synthesized DNA
sequence is shown in SEQ ID NO: 89, and the amino acid sequence encoded
by a coding region contained in the synthesized DNA sequence is shown in
SEQ ID NO: 90. The plasmid was designated as pCR2.1-iPDH.

Construction of pDI626PGK-T-iPDH

[0177] pCR2.1-iPDH was digested with restriction enzymes Sac II and Sal I
to excise a 1080-bp fragment. The resultant was ligated to a pDI626PGK-T
vector digested with restriction enzymes Sac II and Sal I in a similar
manner. The obtained sequence was sequenced, thereby confirming that a
plasmid of interest had been constructed. The thus obtained plasmid was
designated as pDI626PGK-T-iPDH.

Construction of pENT-ADC

[0178] PCR was performed using pDI626-ADC as a template and the following
primers.

[0180] The thus obtained 1809-bp PCR product was introduced into a
pDONR221 P1-P4 donor vector by gateway BP reaction. The obtained clone
was sequenced, thereby confirming that no mutation was present in any
part of the nucleotide sequence of the insert. The thus obtained plasmid
was designated as pENT-ADC.

Construction of pENT-CTFA

[0181] PCR was performed using pDI626PGK-CTFA as a template and the
following primers.

[0182] The obtained 1823-bp PCR product was introduced into a pDONR221
P4r-P3r donor vector by gateway BP reaction. The obtained clone was
sequenced, thereby confirming that no mutation site was present in any
part of the nucleotide sequence of the insert. The thus obtained plasmid
was designated as pENT-CTFA.

Construction of pDI626-CTFB

[0183] pT7Blue-CTFB was digested with restriction enzymes BamH I and Sal I
to excise a 771-bp fragment. The resultant was ligated to a pDI626 vector
digested with restriction enzymes BamH I and Sal I in a similar manner.
The obtained sequence was sequenced, thereby confirming that a plasmid of
interest had been constructed. The thus obtained plasmid was designated
as pDI626-CTFB(+A).

[0184] PCR was performed under the following conditions using the
following primers in order to correct mutation sites in primers.

[0189] After purification of the reaction solution using a MinElute PCR
purification kit (QIAGEN), the resultant was digested with restriction
enzymes BamH I and Sal I. Agarose gel electrophoresis was performed to
excise a 702-bp fragment and then it was purified using a MinElute Gel
extraction kit (QIAGEN). The resultant was ligated to a pDI626 vector
digested with restriction enzymes BamH I and Sal I. The thus obtained
sequence was sequenced, thereby confirming that mutation sites had been
corrected. The thus obtained plasmid was designated as pDI626-CTFB.

Construction of pENT-CTFB

[0190] PCR was performed using pDI626-CTFB as a template and the following
primers.

[0191] The thus obtained 1737-bp PCR product was introduced into a
pDONR221 P3-P2 donor vector by gateway BP reaction. The obtained clone
was sequenced, thereby confirming that no mutation site was present in
any part of the nucleotide sequence of the insert. The thus obtained
plasmid was designated as pENT-CTFB.

[0198] After purification of the reaction solution using a MinElute PCR
purification kit (QIAGEN), the resultant was digested with restriction
enzymes Sac I and Kpn I. Agarose gel electrophoresis was performed to
excise a 1717-bp fragment. After purification using a MinElute Gel
extraction kit (QIAGEN), the resultant was ligated to the pDI626GAP
vector (APP. Env. Micro., 2009, 5536) digested with restriction enzymes
Sac I and Kpn I. The obtained sequence was sequenced, thereby confirming
that a plasmid of interest had been constructed.

Construction of pEXP(Ura)-ADC-CTFA-CTFB

[0199] The 3 obtained entry clones (pENT-ADC, pENT-CTFA, and pENT-CTFB)
were incorporated into a pDEST626(2008) expression vector by Gateway LR
reaction. The thus obtained clones were confirmed by PCR for insert size,
thereby confirming correct recombination. Sequencing was performed,
thereby confirming that no error was present in the sequence. The thus
obtained plasmid was designated as pEXP(Ura)-ADC-CTFA-CTFB.

Preparation of #3-17 Strain

[0200] The pEXP(Ura)-ADC-CTFA-CTFB expression vector was cleaved and
linearized with restriction enzymes Aat II and BssH II. After ethanol
precipitation, the resultant was dissolved in 0.1× TE Buffer and
then Saccharomyces cerevisiae YPH499 (Stratagene) was transformed using a
Frozen EZ yeast transformation kit (Zymoresearch). The obtained clones
were subjected to colony PCR, thereby confirming 25 clones into which
adc, ctfA, and ctfB genes had been introduced. The strain with the
highest acetone production amount was designated as #3-17.

Preparation of #15-10 Strain

[0201] The pDI626PGK-T-iPDH expression vector of the ipdh gene that was a
synthetic gene expected to convert acetone to isopropanol was cleaved and
linearized with restriction enzymes Aat II and BssH II. After ethanol
precipitation, the resultant was dissolved in 0.1× TE Buffer, and
then the #3-17 acetone-producing yeast was transformed using a Frozen EZ
yeast transformation kit (Zymoresearch). The thus obtained 14 clones were
subjected to colony PCR, thereby confirming 13 clones in which the ipdh
gene had been introduced. The strain with the highest isopropanol
production amount was designated as #15-10.